Phosphatases, enzymes that remove a phosphate group from amino-acid substrates, can be subdivided according to their substrate specificity. Myriad evidence has demonstrated that protein phosphatase 2A (PP2A), a family of serine/threonine-specific (Ser/Thr) phosphatases, regulates many, if not most, aspects of cellular activities and is a critical tumor suppressor. A team at the University of Washington recently determined the first crystal structure of a PP2A holoenzyme (a form sufficient for full catalytic activity) composed of three different subunits (i.e., a heterotrimer). Their structure provides a foundation for understanding PP2A regulation, satisfactory mechanistic explanations for human tumorigenic mutations, and the structural basis for understanding PP2A substrate recruitment and specificity, a critical issue, given the high number of PP2A substrates.

Overall structure of the heterotrimeric PP2A holoenzyme. "Front" (left) and "top" (right) views show the scaffold Aα subunit (blue), catalytic Cα subunit (orange), and regulatory B56γ1 subunit (green). The red arrow in the "front" view points to the position of the active site in the Ca subunit with two metal ions (purple). Microcystin-LR, a PP2A-specific inhibitor (regulator), is shown in stick representation. The "top" view shows that the C-terminal tail of the C subunit resides on the interface between the A and B subunits where it can stabilize the A–B interaction. The overall size of the trimeric complex is about 90 Å x 90 Å x 70 Å.

Regulating Cellular Activities

Each of the myriad activities making up the daily life of a cell comprises a carefully choreographed dance of biochemical reactions, each one starting and stopping, speeding up and slowing down on cue, every minute of every day for as long as we live. The signaling processes, such as signal transduction, necessary to regulate this incessant activity in response to changing conditions within the cell are themselves well-ordered sequences of reactions. A process in which molecular fragments called phosphate groups are controllably added to and removed from (reversible protein phosphorylation) proteins by other proteins known as enzymes is a fundamental mechanism for the regulation of cellular activities.

One of these enzymes, protein phosphatase 2A (PP2A), is an exceptionally important player with a thick pile of evidence demonstrating that it not only regulates many, if not most, aspects of cellular activities but is also a critical tumor suppressor. Deregulation of PP2A (so that the phosphate groups are not removed in needed situations) is associated with multiple human cancers, Alzheimer’s Disease, and increased susceptibility to pathogen infections. To shed light on the details of how PP2A works, Cho and Xu have determined the first crystal structure of the enzyme. Their structure answers many but not all questions regarding PP2A regulation and sets the stage for investigation at a more detailed level.

Recent work has shown that many phosphatases are highly regulated, largely through the formation in the right place and right time of active protein phosphatase complexes with different regulatory or targeting subunits. Together with its cousin PP1, PP2A accounts for greater than 90% of Ser/Thr phosphatase activity in most tissues and cells. Deregulation of PP2A is associated with multiple human cancers, Alzheimer’s Disease, and increased susceptibility to pathogen infections. A typical PP2A holoenzyme contains a scaffold A subunit, a catalytic C subunit, and one of many possible (at least 18 in humans) regulatory B subunits, which are divided into B, B’, B” and B’” families. The formation of an ABC heterotrimeric PP2A holoenzyme is in part controlled by the methylation of the C-terminal carboxyl group at one end of the catalytic subunit.

After years of painstaking struggle, using data collected at ALS Beamlines 5.0.2 and 8.2.2, the Washington team determined the first crystal structure of the PP2A heterotrimeric holoenzyme containing Aα, Cα, and B56γ1 subunits as well as an inhibitor protein. The structure revealed the overall domain organization of PP2A holoenzyme and all-important interfaces that determine the assembly of a functional PP2A holoenzyme, and therefore provided a foundation for understanding PP2A regulation by PP2A post-transcriptional modifications (e.g., phosphorylation and methylation) and the binding of PP2A regulatory proteins. For example, functional assembly of an ABC heterotrimeric PP2A holoenzyme is regulated by the methylation of the C-terminal carboxyl group at one end of the catalytic subunit.

Structural details revealed include 15 modules comprising multiple amino acids called HEAT repeats that form a horseshoe-shaped scaffold in the PP2A A subunit and use one side of its ridge to hold the catalytic C and regulatory B56γ1(B’) subunits together. The structure also showed that the C-terminal tail of the C subunit resides on the interface between the A and B subunits and thus stabilizes the A–B interaction. Strikingly, the methylated C-terminal carboxyl group is docked in a highly negatively charged groove. Once demethylated, there would be charge–charge repulsion between this groove and the negative charge in the C-terminal carboxyl group. It is likely that the methylation of the C-terminal carboxyl group regulates PP2A assembly by simply neutralizing charge repulsion.

In addition, the team's structure also provided satisfactory mechanistic explanations for human tumorigenic mutations, which are mostly found in the inter-subunit interfaces in the structure. Regarding PP2A substrate recruitment and specificity, the structure of the PP2A holoenzyme shows that the active site is located in the middle of a large open surface, with the majority of this surface provided by the large concave surface of the B’ regulatory subunit. Therefore PP2A substrates can be recruited to the active site from various surfaces or directions. The structure thus reveals potential surface areas that may participate in substrate recognition.

In sum, the Washington team's structure of the PP2A holoenzyme does not answer all questions regarding PP2A regulation, but it does allow testing such questions at the next level.

Research conducted by U.S. Cho and W. Xu (University of Washington).

Research Funding: Burroughs Welcome Fund and the Keck Center for Pathogenesis at the University of Washington. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (BES).